Working with billions of tiny cells can pose a problem when you need to count the total number of cells in a sample. Fortunately, through precise serial dilution of a sample, it is possible to get down to a number that is much easier to work with.

## Huge Micro Problem

As you know, bacteria are everywhere, invisible to the naked eye, yet influencing every environment on Earth. What happens when you need to know how many individual bacterial cells are contaminating a food, living in an environmental sample, or growing in a culture tube? You need some method for counting the bacteria accurately. But, it is not uncommon for a liquid culture of bacteria to have a billion cells in every milliliter of media.

Think about that for one second. In your kitchen, you probably have a teaspoon. Every teaspoon has about 5 milliliters. That means that every teaspoon of liquid could potentially have 5 billion bacteria in it. Even if you counted one bacteria every second, it would take you over 150 years to get to 5 billion! Obviously, this is not a viable option. So, what can you do? You need fewer bacteria to count.

Ideally, you want to only have to count between 30 and 300 bacteria, a range of numbers that takes only at most a few minutes to count. But, how do we get there?

## Serial Dilution

The answer is through dilution. If you simply pull out a smaller, exact quantity of culture liquid, you could count those bacteria and, based on how much you pulled out of the total, you can determine how many bacteria are in your original sample.

Sounds easy, right?But first, one more analogy: you have billions of bacterial cells and need to get down to 30 to 300. In order to do that, you would have to dilute your sample about 10 million-fold. To do this, you would need to take about 15 milliliters of your sample, about 3 teaspoons, and dilute it into your swimming pool! I doubt this is a viable option, especially if you’re working in a cramped lab space. So instead, let’s not dilute just once.

We can dilute once, then dilute this dilution, only to dilute this dilution, and so on until we get to the appropriate concentration of cells. This is called a serial dilution.A **serial dilution** is a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration. Each dilution will reduce the concentration of bacteria by a specific amount.

So, by calculating the total dilution over the entire series, it is possible to know how many bacteria you started with. The best way to fully grasp serial dilutions is to try out the procedure yourself.

## How to Perform a Serial Dilution

I’m going to walk you through an example serial dilution using the easiest method, but, once you grasp the concept, you can change the actual numbers to whatever works best for you and do it the same way.To start, we need 10 milliliters (10 ml) of your original bacterial culture (labeled OBC). Before we start diluting, we need to prepare several dilution blanks, which are tubes containing your diluting liquid in exact quantities.

Your liquid could be growth media, saline, sterile water, or any other appropriate liquid. For this example, we need 5 dilution blanks, numbered 1-5. In each tube, we need exactly 9 ml of liquid media.

The reason we need 9 ml will become apparent soon.The tubes should be lined up like this:

The first step is to gently shake or swirl the tube. This will ensure that your cells are evenly distributed in the tube.

If your cells settle to the bottom, and you remove liquid without swirling, you run the risk of not getting enough cells, invalidating your final count. Remember to always swirl the tube before removing liquid.Once swirled, carefully transfer exactly 1 ml from your OBC Tube to Tube 1. Now, you should have 10 ml of liquid in Tube 1.

Exactly one-tenth of your cells are now in a new tube with a final volume of 10 ml. You just performed a 1 in 10 dilution, or it could be written 1/10. 1 is the volume you transferred, and 10 is the final volume of the tube after the transfer.

Now, you are done with tube OBC, and Tube 1 becomes the next tube to be diluted. Like we did before, swirl your tube before transferring 1 ml from Tube 1 into Tube 2. Again, exactly one-tenth of your cells in Tube 1 are transferred to Tube 2, with a final volume of 10 ml.

Tube 1 should have exactly 9 ml left. Tube 2 now contains a 1 in 10 dilution of Tube 1. In order to calculate the total dilution from Tube OBC, simply multiply your two dilutions: 1/10 X 1/10 = 1/100. So far, you have performed a 1/100 dilution from the original bacterial culture.

You want to follow the same procedure for the remaining dilution blanks: 1 ml from Tube 2 is transferred to Tube 3; 1 ml from Tube 3 is transferred to Tube 4; and, finally, 1 ml from Tube 4 is transferred to Tube 5. Each transfer is another 1 in 10 dilution. To calculate the final dilution, simply multiply all the dilutions together: 1/10 X 1/10 X 1/10 X 1/10 X 1/10 = 1/100,000.Now we might have a reasonable number of cells to count. To finish the technique, let’s imagine we count the cells in Tube 5 and find 50 total cells, right in the desired 30 to 300 zone. In order to determine how many cells we started with in our original culture, all you need to do is multiply the cell count by the total dilution: 50 X 100,000 = 5,000,000 bacterial cells in our original 10 ml sample.

I bet you would rather count to 50 than 5 million!It is important to note that you can use any volumes here. If your dilution blanks are 6 ml, and you are transferring 1 ml, the dilution would be 1/7. The math is exactly the same: 1/7 X 1/7 X 1/7 X 1/7 = 1/2401.

You also don’t need to have the same final volume in every tube. You can dilute 1/2, then 1/5, then 1/8. Simply multiply as before to get the final dilution: 1/2 X 1/5 X 1/8 = 1/80.Now you might be wondering how we count the tiny cells. There are several methods that could be used, but we will use the culture plate method. We can take a sample of Tube 5 and grow the cells on a culture plate. Then, once the bacteria start to grow, they form colonies that eventually get large enough to see.

Then, we can count the colonies and back calculate to find the original concentration of bacteria in our sample. Congratulations! You have solved what started out to be a pretty daunting problem!

## Lesson Summary

Let’s briefly review serial dilutions.A **serial dilution** is a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration.

The easiest method is to make a series of 1 in 10 dilutions. In this method, exactly 1 ml of each successive dilution is transferred into exactly 9 ml of liquid in a dilution blank, creating a 1/10 dilution. In order to calculate the final dilution, all you need to do is multiply the 1/10 X 1/10, continuing like this for each step in the dilution.

To determine exactly how many cells you started with, simply take the number of colonies you counted, and multiply it by your final dilution factor.

## Learning Outcomes

Once you’ve completed this lesson, you may be able to:

- Create a serial dilution and recognize its purpose
- Identify the steps necessary for developing a serial dilution
- Know how to count bacteria cells